Japanese Journal of Clinical Oncology 34:184-190 (2004)
© 2004 Foundation for Promotion of Cancer Research
Serum Vascular Endothelial Growth Factor per Platelet Count in Hepatocellular Carcinoma: Correlations with Clinical Parameters and Survival
Division of Oncology and Hematology, Department of Internal Medicine, Korea University Medical Center, Seoul, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Background: Platelets have been reported to act as transporters of tumor-originated vascular endothelial growth factor (VEGF), contributing to tumor angiogenesis and progression. Serum VEGF per platelet count, as an indirect theoretical estimate of VEGF in platelets, may predict the malignant potential of tumors. However, its prognostic significance is still unclear in hepatocellular carcinoma (HCC), a highly vascular tumor.
Methods: Serum VEGF was measured by enzyme-linked immunosorbent assay. We compared serum VEGF, platelet count and serum VEGF per platelet count in 52 HCC patients, 26 liver cirrhosis patients and 30 healthy controls. The relation of serum VEGF per platelet count with clinicopathologic variables of HCC patients and the prognostic significance were investigated.
Results: Serum VEGF per platelet count in HCC patients was higher than in liver cirrhosis patients and healthy controls (P < 0.01). There was a statistically significant correlation between serum VEGF and platelet count in HCC patients (r = 0.751, P < 0.01). Serum VEGF per platelet count was higher in patients with advanced stage and portal vein thrombosis (P < 0.01). Patients with high serum VEGF per platelet count (>1.4 pg/106) showed poor response to treatment and shorter overall survival (P < 0.01). Serum VEGF per platelet count was an independent prognostic factor with the presence of portal vein thrombosis (P < 0.01).
Conclusions: Serum VEGF per platelet count could be a feasible prognostic indicator during the follow-up of patients with HCC.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Hepatocellular carcinoma (HCC) is a highly vascular tumor, thus, angiogenesis is considered to be important for its progression (1). Vascular endothelial growth factor (VEGF) is a well known, potent angiogenic factor which enhances vascular permeability, promoting the extravasation of protein to form a stromal matrix and tumor invasion (2). Serum VEGF was reported to predict venous invasion in patients with HCC (3), and researchers have reported correlation between higher serum VEGF and poorer prognosis in several cancers (4–7). However, controversy still exists as to whether serum or plasma VEGF will provide the information about tumor angiogenesis and prognosis, because serum VEGF can be influenced by VEGF released from platelets during clotting (8,9).
It has been reported that the VEGF content of platelets is several-fold higher in cancer patients than in healthy subjects (10,11). Platelets are also known to endocytose and concentrate plasma proteins such as VEGF and later transport them into their granules (12). Thus, some authors have suggested that VEGF secreted from tumor cells could be stored and transported by platelets in the bloodstream, and that this reservoir of VEGF might have a role in tumor angiogenesis and invasion (10,11,13,14). Based on this hypothesis, VEGF load in platelets may indirectly show tumor-originated VEGF and predict tumor angiogenic activity better than serum VEGF. A recent study used serum VEGF per platelet count to correct variation of serum VEGF levels in patients with different platelet counts (15). In that study, serum VEGF per platelet count correlated with advancing stage of colorectal cancer, suggesting its role as a standard measure of circulating VEGF. However, there is still little data regarding the prognostic significance of serum VEGF per platelet count in patients with HCC. In this study, we determined the prognostic implication of serum VEGF per platelet count and its relation with clinical parameters in patients with HCC.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Patients
Fifty-two patients with primary HCC were enrolled between 2001 and 2002. Forty-seven of 52 patients underwent pathologic diagnosis. The remaining five patients could not undergo biopsy because of high bleeding tendency. However, these five patients could be diagnosed as HCC because of their typical findings on abdominal computed tomography and high alpha fetoprotein (
FP) level. They were staged based on the TNM staging of The Liver Cancer Study Group of Japan (LCSGJ) (16). They all had liver cirrhosis as an underlying disorder before they were diagnosed as having HCC. Surgical resection of primary mass was not attempted in the patients with HCC because of their poor operability. In 35 patients, a blood sample was taken before their initial treatment. After that, 20 patients and 15 patients were treated with either transarterial chemoembolization or systemic chemotherapy, respectively, as the first line therapy. The other 17 patients had taken between two and four cycles of transarterial chemoembolization before they were enrolled into this study. In these patients, systemic chemotherapy was started after blood sampling because of the disease progression. Blood was taken when they were in the follow-up period at least 4 weeks after their last cycle of transarterial chemoembolization. Their median follow-up was 5 months (range, 1–14 months) after they were enrolled into the study, and the response to treatment was assessed every two cycles of treatment. For comparison of serum VEGF and platelet count, 30 healthy controls and 26 liver cirrhosis patients were enrolled as control groups. Patients with infection or chronic inflammation were excluded from this study. Healthy controls were volunteers who turned out to have no medical disorders after their health examinations, including liver function test and abdominal ultrasound. The study was performed with informed consent obtained from all patients and control subjects, and was approved by the institutional review board for human investigation at Korea University.
Collection of Samples
Venous blood was drawn from all patients and healthy volunteers using 21-gauge needle. In order to count platelets, blood counts were performed with an automated blood Coulter counter. For preparation of serum samples, the tubes were centrifuged at 3000 g for 10 min at room temperature, 30 min after blood collection. Serum was separated and the aliquots stored at –80°C until assay by enzyme-linked immunosorbent assay (ELISA) of VEGF.
ELISA of VEGF
Serum VEGF concentrations were measured with Biotrak VEGF human ELISA system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Ninety-six-well microtiter plates were coated with anti-human VEGF antibody. Then 50 µl of serum was added to each well and incubated for 2 h at room temperature. The plates were incubated with biotinylated anti-human VEGF165 for 1 h and streptavidin–HRP reagent for 30 min. A color reaction was induced by the addition of premixed TMB substrate solution and was stopped 30 min later by the addition of a stop solution. The absorbency was measured on an ELISA reader at 450 nm. All samples were assayed in duplicate and the mean value was used for VEGF determination. The sensitivity limit of this assay was 8.0 pg/ml for VEGF.
Statistical Analysis
All data are presented as median values with the 25th and 75th interquartile ranges. Comparisons of continuous variables in different subgroups were performed using the Mann–Whitney U test or the Kruskal–Wallis test. Coefficient of correlation (r) between serum VEGF concentrations and platelet counts was calculated using the Spearman’s rank test. Survival curves were calculated using the Kaplan–Meier method and compared by the log-rank test. The Cox proportional hazards regression model was used for multivariate analyses after univariate analysis defined relevant prognostic variables. Statistical significance was defined as P < 0.05. All the statistical analyses were performed using a statistical software package (SPSS, Version 10.0, Inc., Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Serum VEGF, Platelet Count and Serum VEGF per Platelet Count in Patients with HCC, Liver Cirrhosis and Healthy Controls
The basal characteristics of the patients with HCC and liver cirrhosis were similar, including viral marker status and liver function (Table 1), and all the patients with HCC had liver cirrhosis as an underlying disorder. Table 1 demonstrates that serum VEGF level was higher in the patients with HCC compared to those with the liver cirrhosis and healthy controls (P < 0.01). Although platelet count was lower than in healthy subjects, it was also higher in patients with HCC than those with liver cirrhosis (P < 0.01). Serum VEGF per platelet count was calculated by dividing serum VEGF concentration by the platelet count, and HCC patients showed higher values as compared to the healthy controls and liver cirrhosis patients (P < 0.01).
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Correlation of Serum VEGF Level and Platelet Count
In the patients with HCC, there was a statistically significant correlation between serum VEGF level and platelet count (r = 0.75, P < 0.01) (Fig. 1). There was an increase in serum VEGF level according to the increments of platelet count. However, there was no correlation between serum VEGF levels and platelet count in the patients with liver cirrhosis and healthy controls (P = 0.11, 0.52, respectively).
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Correlation between Serum VEGF, Platelet Count and Serum VEGF per Platelet Count and Clinical Features in the Patients with HCC
Serum VEGF level and platelet count were increased with advanced tumor stage and the presence of portal vein thrombosis. Thus, serum VEGF per platelet count was higher in the group at stage III/IV and with portal vein thrombosis than in the group at stage I/II and without portal vein thrombosis (P = 0.03, 0.04, respectively). Serum VEGF level and platelet count were higher in the patients without splenomegaly than those with splenomegaly, but serum VEGF per platelet count was not different with or without splenomegaly. There was no correlation between serum VEGF per platelet count and other clinical features including serum
FP level and child class. In our study, 17 patients had been treated with transarterial chemoembolization before they were enrolled, so we compared the serum VEGF level and platelet count of the treatment-naive 35 patients with the 17 patients who had been treated with transarterial chemoembolization. There was no difference between the two groups (P > 0.05) (Table 2).
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Impact of Serum VEGF per Platelet Count on Response and Survival
We assessed the relationship of serum VEGF per platelet count with the treatment response and overall survival. The group with high serum VEGF per platelet count (>1.4 pg/106) and that with low serum VEGF per platelet count (
1.4 pg/106) were divided by the median value. In patients treated with transarterial chemoembolization and systemic chemotherapy, the number of cases showing disease progression was higher in the group with high serum VEGF per platelet count (Table 3). The high serum VEGF per platelet count group was inferior to the low serum VEGF per platelet count group in terms of survival duration (P = 0.01) (Fig. 2). Thirty-three patients died during the follow-up period and the causes of death were as follows: disease progression (n = 10), hepatic failure (n = 13) and combined (n = 10). In the multivariate analysis including clinical variables, serum VEGF per platelet count was found to be an independent prognostic factor with portal vein thrombosis in HCC patients (Table 4). High serum VEGF per platelet count (>1.4 pg/106) was significantly related with worse survival in patients with and without portal vein thrombus (P < 0.01, both) (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Tissue expression of VEGF and its correlation with prognosis have been investigated by immunohistochemistry (17,18). However, immunohistochemistry has a limitation, because it requires a tumor specimen. On the other hand, the measurement of VEGF in blood does not require a tumor specimen, thus it is applicable to every cancer patient. On the other hand, serum VEGF level can be influenced by platelet count and it is still unclear whether serum VEGF level can reflect the actual events occurring at the tumor site.
The role of platelets in tumor angiogenesis and prognosis has been well described. Thrombocytosis has been reported to be a direct cause of poorer survival in patients with lung and endometrial cancer (19,20) and a direct correlation between platelet counts and serum VEGF has also been reported (11,15,21). In addition, platelets have been reported to endocytose and store various angiogenic factors such as VEGF and platelet-derived growth factor (PDGF) in their granules, and these molecules are secreted immediately after platelet activation (22). Therefore, platelets may transport VEGF to its target cells, restricting the angiogenic activity of circulating VEGF to sites where coagulation takes place, such as a healing wound (23). This view is supported by the fact that platelet aggregation with cancer cells is commonly observed during the process of angiogenesis and metastasis (24). This function of platelets may explain the lack of apparent angiogenesis observed outside of the tumor in patients with high amounts of circulating VEGF.
In this study, HCC patients showed higher serum VEGF and lower platelet count than healthy controls (P < 0.01). In addition, serum VEGF level was significantly correlated with platelet counts in HCC patients (r = 0.751, P < 0.01), but there was no such correlation in liver cirrhosis patients and healthy controls, in agreement with the previous studies (11,15). These facts would imply the abundance of tumor-originated VEGF in platelets of the HCC patients.
In both groups with transarterial chemoembolization and systemic chemotherapy, the number of cases with disease progression was higher in the patients with high serum VEGF per platelet count (>1.4 pg/106). Serum VEGF per platelet count was also increased with tumor size, advanced TNM stage and the presence of portal vein thrombosis. In addition, those 10 patients with distant metastasis at the time of diagnosis showed high serum VEGF per platelet count. Based on these findings, we suggest that serum VEGF per platelet count predicts tumor aggressiveness and treatment outcome.
Serum VEGF per platelet count was higher in the patients with HCC than in those with liver cirrhosis. The presence of HCC could contribute to this difference, because both groups had liver cirrhosis and similar characteristics such as liver function and viral markers. This may suggest the possible role of serum VEGF per platelet count as an indicator of the development of HCC in patients with liver cirrhosis during follow-up.
In the multivariate survival analysis, high serum VEGF per platelet count (>1.4 pg/106) and portal vein thrombosis were independent prognostic indicators for overall survival. This suggests that serum VEGF per platelet count could be a better prognostic factor than serum VEGF itself. Child–Pugh class failed to show a prognostic significance in this study, but it does not imply that the prognosis of HCC is independent of underlying liver functions. We suggest that it is related to the relatively small size of the patient population of this study.
Basically, serum VEGF is a combination of both VEGF released from platelets on coagulation and the circulating plasma VEGF. Furthermore, platelets may normally contain a certain amount of VEGF in their granules regardless of the presence of tumor, and this, therefore, needs to be taken into account. However, because serum VEGF per platelet count in healthy controls was very low compared to that in HCC patients in this study, and since plasma VEGF level has been known to be very low compared to serum, the impact of these factors should be small in this study.
In conclusion, this study demonstrates that serum VEGF per platelet is a reliable indicator of prognosis in patients with HCC. Therefore, serum VEGF per platelet count could be a useful clinical parameter for follow-up and establishing treatment strategy. Further study with a larger patient population and longer duration is warranted to clarify its value in clinical application.
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Acknowledgment |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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This study was supported by a grant from Korea University Medical Science Research Center and Brain Korea 21 project.
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FOOTNOTES |
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+ For reprints and all correspondence: Byung Soo Kim, Division of Oncology and Hematology, Department of Internal Medicine, Anam Hospital, Korea University Medical Center, 126-1, Anam-dong 5-ga, Sungbuk-ku, Seoul 136-705, Korea. E-mail: kbs0309{at}ns.kumc.or.kr
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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1 Li XM, Tang ZY, Liu YK, Ye SL. Significance of vascular endothelial growth factor mRNA expression in invasion and metastasis in hepatocellular carcinoma. J Exp Clin Cancer Res 1998;17:13–7.[Web of Science][Medline]
2 Dvorak HF, Brown IF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hypermeability, and angiogenesis. Am J Pathol 1995;146:1029–39.[Abstract]
3 Poon RT, Ng IO, Lau C, Zhu LX, Yu WC, Lo CM, et al. Serum vascular endothelial growth factor predicts venous invasion in hepatocellular carcinoma: a prospective study. Ann Surg 2001;233:227–35.[CrossRef][Web of Science][Medline]
4 Salven P, Ruotsalainen T, Mattson K, Joensuu H. High pre-treatment serum level of vascular endothelial growth factor (VEGF) is associated with poor outcome in small-cell lung cancer. Int J Cancer 1998;79:144–6.[CrossRef][Web of Science][Medline]
5 O’Byrne JK, Dobbs N, Propper D, Smith K, Harris AL. Vascular endothelial growth factor, platelet counts, and prognosis in renal cancer. Lancet 1999;353:1494–5.[Web of Science][Medline]
6 Akbulut H, Altuntas F, Akbulut KG, Ozturk G, Cindoruk M, Unal E, et al. Prognostic role of serum vascular endothelial growth factor, basic fibroblast growth factor and nitric oxide in patients with colorectal carcinoma. Cytokine 2002;20:184–90.[CrossRef][Web of Science][Medline]
7 Karayiannakis AJ, Bolanaki H, Syrigos KN, Asimakopoulos B, Polychronidis A, Anagnostoulis S, et al. Serum vascular endothelial growth factor levels in pancreatic cancer patients correlate with advanced and metastatic disease and poor prognosis. Cancer Lett 2003;194:119–24.[CrossRef][Web of Science][Medline]
8 Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, Walters C, et al. Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer 1998;77:956–64.[Web of Science][Medline]
9 Webb NJA, Bottomley MJ, Watson CJ, Brenchley PEC. Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. Clin Sci 1998;94:395–404.[Medline]
10 Vermeulen PB, Salven P, Benoy I, Gasparini G, Dirix LY. Blood platelets and serum VEGF in cancer patients. Br J Cancer 1999;79:370–6.[Web of Science][Medline]
11 Salgado R, Vermeulen PB, Benoy I, Weytjens R, Huget P, Van Marck E, et al. Platelet number and interleukin-6 correlate with VEGF but not with bFGF serum levels of advanced cancer patients. Br J Cancer 1999;80:892–7.[CrossRef][Web of Science][Medline]
12 Handagama PJ, George JN, Shuman MA, McEver RP, Bainton DF. Incorporation of a circulating protein into megakaryocyte and platelet granules. Proc Natl Acad Sci USA 1987;84:861–5.
13 Salven P, Orpana A, Joensuu H. Leukocytes and platelets with cancer contain high levels of vascular endothelial growth factor. Clin Cancer Res 1999;5:487–91.
14 Benoy I, Salgado R, Colpaert C, Weytjens R, Vermeulen PB, Dirix LY. Serum interleukin 6, plasma VEGF, serum VEGF, and VEGF platelet load in breast cancer patients. Clin Breast Cancer 2002;2:311–5.[Medline]
15 George ML, Eccles SA, Tutton MG, Abulafi AM, Swift RI. Correlation of plasma and serum vascular endothelial growth factor levels with platelet count in colorectal cancer: Clinical evidence of platelet scavenging? Clin Cancer Res 2000;6:3147–52.
16 Liver Cancer Study Group of Japan. General Rules for the Clinical and Pathological Study of Primary Liver Cancer, 2nd edn. Tokyo: Kanehara 2003; 23.
17 Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlation with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 1995;55:3964–8.
18 Amaya H, Tanigawa N, Lu C, Matsumura M, Shimomatsuya T, Horiuchi T, et al. Association of vascular endothelial growth factor expression with tumor angiogenesis, survival and thymidine phosphorylase/platelet-derived endothelial cell growth factor expression in human colorectal cancer. Cancer Lett 1997;119:227–35.[CrossRef][Web of Science][Medline]
19 Cox G, Walker RA, Andi A, Steward WP, O’Bryne KJ. Prognostic significance of platelet and microvessel counts in operable non-small cell lung cancer. Lung Cancer 2000;29:169–77.[Web of Science][Medline]
20 Gücer F, Moser F, Tamussino K, Reich O, Haas J, Arikan G, et al. Thrombocytosis as a prognostic factor in endometrial carcinoma. Gynecol Oncol 1998;70:210–4.[CrossRef][Web of Science][Medline]
21 Verheul HM, Hoekman K, Luykx-de Bakker S, Eekman CA, Folman CC, Broxterman HJ, et al. Platelet: Transporter of vascular endothelial growth factor. Clin Cancer Res 1997;3:2187–90.
22 Harrison P, Wilbourn B, Debil N, Vainchenker W, Breton-Gorius J, Lawrie AS, et al. Uptake of plasma fibrinogen into granules of human megakaryocytes and platelets. J Clin Invest 1989;84:1320–4.[Web of Science][Medline]
23 Maloney JP, Silliman CC, Ambruso DR, Wang J, Tuder RM, Voelkel NF, et al. In vitro release of vascular endothelial growth factor during platelet aggregation. Am J Physiol 1995;275:1054–61.
24 Trikha M, Nakada MT. Platelets and cancer: Implications for antiangiogenic therapy. Semin Thromb Hemos 2002;28:39–44.
Received December 24, 2003; accepted March 2, 2004
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